In order for milk to coagulate and eventually form cheese, enzymes must be added to breakdown the proteins that keep milk a liquid. More particularly, when proteins are denatured or otherwise modified, milk loses its liquid structure and begins to coagulate. Rennets, milk-coagulating enzymes traditionally obtained from the abomasum (the fourth stomach of the calf) have long been used in cheese making. The main enzyme in calf rennet is chymosin.
Calf-rennet, however, is expensive and has increasingly been replaced with rennet derived from microorganisms. U.S. Pat. No. 4,526,792 discloses the use of R. miehei as microbial rennet in the production of cheese. (All documents cited are, in relevant, part, incorporated herein by reference.) R. miehei does not contain chymosin, but instead acid proteases, which are similar in function to chymosin.
Among the commercially-available R. miehei extracts, several possess some degree of enzymatic activity, principally from acid proteases. An extract substantially devoid of acid-protease activity can, however, be obtained by the removal of enzymatic activity by a number of well-known biochemical processes. The resulting non-enzymatic milieu is substantially devoid of enzymatic activity. A number of methods well-known to those skilled in the art to remove enzymatic activity are known and include affinity gel column chromatography and subsequent elution of the adsorbed microbial rennet. See, e.g., Kobayashi, et al., “Rapid isolation of microbial milk-clotting enzymes by N-acetyl-(or N-isobutyryl)-pepstatin-aminohexylagarose” Anal. Biochem., 122: 308-312 (1982) (microbial rennet from R. miehei purified by use of affinity gel column using N-acetylpepstatin as affinity ligand). Enzymes can also be separated on affinity gel columns using Cibacron Blue F3GA (“CB”). See, e.g., Dean, et al., “Protein purification using immobilized triazine dyes,” J. Chromatogr., 165: 301-319 (1979) and Burgett, et al., “Cibacron Blue F3GA affinity chromatography”, Am. Lab., 9(5): 74, 78-83 (1977) (describing separation of enzymes on CB columns, including for example, kinases and nucleases). U.S. Pat. No. 4,743,551 describes the use of a blue dye affinity ligand and elution of the adsorbed rennet to produce a purified R. miehei rennet. A proteinaceous extract of R. miehei substantially devoid of acid-protease activity can also obtained by other techniques well-known to those of skill in the art, including thermal inactivation and molecular weight sieve.
There exists a delicate homeostatic balance between the rates of synthesis and degradation in the skin cells and underlying connective tissues including collagen and elastin, the principal structural proteins in mammalian skin. This balance allows the cells and tissues to regenerate as well as repair and replace damaged cells and tissues caused by environmental stressors. Thus, in the case of collagen, both endogenous and exogenous signals regulate the transcription of collagen mRNA and its subsequent translation into non-structural collagen subunits known as procollagen. More particularly, procollagen undergoes post-translational modification, including hydroxylation of proline and lysine residues to hydroxyproline and hydroxylysine. Procollagen is susceptible to degradation by collagenases, including matrix metalloproteinases. After being secreted through Golgi apparatus, procollagen is further processed into collagen via proteolytic removal of noncollagenous portions of the polypeptide (i.e., by proteases). The collagen molecules are then assembled into mature collagen fibrils which, in turn are cross-linked, and are more resistant to metalloproteases.
The visible signs of aging (e.g., fine lines and wrinkles) are correlated with a decrease in the level of collagen in the skin. This is can be attributed both to decreased synthesis as well as increased enzymatic degradation by collagenases, in particular Collagenase I also known as Matrix Metalloprotease 1 (MMP1). The degradative activity of MMP1 is regulated by the concentration of an endogenous protease inhibitor, Tissue Inhibitor of Matrix Metalloprotease-1 (TIMP1).
Prior art compositions have attempted to increase collagen synthesis in the skin by a number of molecular mechanisms. See, e.g., U.S. Pat. No. 6,846,812. KTTKS is a pentapeptide is derived from a fragment of the C-terminal portion of Collagen I. It has been studied in detail by Katayama et al., “A pentapeptide from type I procollagen promotes extracellular matrix production,” J. Biol. Chem., 268(14): 9941-9944 (1993).
Another approach to reducing the appearance of the signs of aging has been to enhance epidermal cell turnover by applying exfoliants. Two widely-used classes of exfoliants well-known to those of skill in the art are acid proteases (e.g., Cathepsin D-like), see, e.g., U.S. Pat. Nos. 6,656,701 and 6,569,437, and hydroxycarboxylic acids (e.g., alpha-hydroxy acids, such as glycolic acid).
In a poster presented at the February 2005 annual meeting of the American Academy of Dermatology in New Orleans, Leyden et al. described the use of a botanical extract from R. miehei that has Cathepsin-D like activity to enhance epidermal cell renewal (i.e., exfoliation) and thereby improve the appearance of environmentally damaged skin. Suprisingly, the proteinaceous extract of R. miehei of the present invention that is substantially devoid of acid protease activity, reduces the appearance of the signs of aging by a primary mode of action that is not based on exfoliation.
Thus, there remains a long-felt but as yet unmet need to increase Collagen I by upregulating the gene(s) that codes for the synthesis of Collagen I, and/or decrease levels of MMP1, either by upregulating the gene(s) that codes for TIMP1 or downregulating the gene(s) that codes for the expression of MMP1. These needs are met by the proteinaceous extract of the present invention. Surprisingly, the extract of R. miehei of the present invention that is substantially devoid of acid protease activity upregulates expression not only of the aforementioned genes, but also notably upregulates the expression of genes that code for fibronectin and vimentin (extracellular matrix glycoproteins involved in cell adhesion, differentiation, and migration) as well as procollagen-lysine 2-oxoglutarate 5-dioxygenase (an enzyme involved in the crosslinking of procollagen to form mature bundled collagen fibers).